1. Field of the Invention
The present invention relates to a thermostable trehalose-releasing enzyme, and its preparation and uses, more particularly, to a novel thermostable trehalose-releasing enzyme which specifically hydrolyses the linkage between a trehalose moiety and the remaining glycosyl moiety in non-reducing saccharides having a trehalose structure as an end unit and having a glucose polymerization degree of 3 or higher, and to the preparation of the enzyme. The present invention further relates to trehalose obtainable by using the enzyme and to compositions containing the same.
2. Description of the Prior
Trehalose or .alpha., .alpha.-trehalose is known as a non-reducing saccharide consisting of glucose units. As is described in Advances in Carbohydrate Chemistry, Vol.18, pp.201-225 (1963), published by Academic Press, USA, and Applied and Environmental Microbiology, Vol.56, pp.3,213-3,215 (1990), trehalose widely exists in microorganisms, mushrooms, insects, etc., though the content is relatively low. Trehalose is a non-reducing saccharide, so that it neither reacts with substances containing amino groups such as amino acids and proteins, induces the amino-carbonyl reaction, nor deteriorates amino acid-containing substances. Thus, trehalose is expected to be used without fear of causing an unsatisfactory browning and deterioration. Because of these, the establishment of the industrial-scale preparation of trehalose has been in great demand.
Conventional preparations of trehalose are, for example, those which are disclosed in Japanese Patent Laid-Open No.154,485/75 wherein microorganisms are utilized, and reported in Japanese Patent Laid-Open No.216,695/83 wherein maltose is converted into trehalose by using maltose- and trehalose-phosphorylases in combination. The former, however, is not suitable for industrial-scale preparation because the content of trehalose contained in microorganisms used as a starting material is usually lower than 15 w/w % (the wording "w/w %" will be abbreviated as "%" in the specification, unless otherwise specified), on a dry solid basis (d.s.b.), and the extraction and purification steps are complicated. The latter has the following demerits: (i) Since trehalose is formed via glucose-1-phosphate, the concentration of maltose as a substrate could not be set to a desired level; (ii) the enzymatic reaction systems of the phosphorylases are reversible reactions, and their yields of the objective trehalose are relatively low; and (iii) it is substantially difficult to retain their reaction systems stably and to continue their enzymatic reactions smoothly. Thus, these conventional preparations have not been actually used as an industrial-scale preparation.
Considering the aforementioned circumstances, the present inventors have energetically studied enzymes which are capable of forming saccharides having a trehalose structure when allowed to act on starch hydrolysates. As a result, the present inventors found that Rhizobium sp. M-11 or Arthrobactor sp. Q36 is capable of producing a novel non-reducing saccharide-forming enzyme which forms non-reducing saccharides having a trehalose structure as an end unit when allowed to act on reducing partial starch hydrolysates having a degree of glucose polymerization of 3 or higher, and simultaneously found that a trehalose-releasing enzyme produced by Rhizobium sp. M-11 or Arthrobactor sp. Q36 can hydrolyse the non-reducing saccharides into trehalose and glucose and/or maltooligosaccharide at a constant amount. These enzymes realized that an objective amount of trehalose can be readily obtained by using starch as a material, and the aforementioned object concerning a trehalose is expected to be attainable.
Enzymes derived from Rhizobium sp. M-11 or Arthrobactor sp. Q36, however, are relatively-low in thermal stability. Thus, in case that these enzymes are utilized for preparing trehalose and non-reducing saccharides having a trehalose structure as an end unit, it is necessary to allow the enzymes to act on at a temperature of below 55.degree. C. With regard to the temperature of enzymatic reaction, as described in the column titled "Enzymes related to saccharides" in the chapter titled "Enzymes related to saccharides and their applications" in "Koso-Ouyou-no-Chishiki" (Knowledge on Enzyme Applications), the first edition, pp.80-129 (1986) that "In the conditions of industrial-scale enzymatic reactions for saccharification, the reactions at a temperature of below 55.degree. C. involves a risk of contamination and a decrease of pH during the reaction, in long-time enzymatic reactions using starch as a material, when an enzyme is allowed to act on at a temperature of below 55.degree. C., because of contamination and a decrease of pH of reaction mixtures which inactivate the activity of such enzymes, and it is necessary to add lysozyme for the prevention of contamination and the pH control of the reaction mixtures. In addition, when the hydrolysis of partial starch hydrolysates is relatively low, insoluble substances may be formed due to retrogradation of starch.
On the other hand, since a thermostable enzyme can maintain its activity at a relatively-high temperature, contamination during the enzymatic reaction is less of a concern and the retrogradation of partial starch hydrolysates is scarcely caused. As a source of thermostable enzymes, thermophilic microorganisms can be generally considered. Regarding a preparation of trehalose using thermophilic microorganisms, as described in Biotechnology Letters, Vol.12, pp.431-432 (1990) and Biotech Forum Europe, Vol.8, pp.201-203 (1991), it was reported that the partially purified enzyme preparation obtainable from the cell and cell extract of Sulfolobus solfataricus (ATCC 49155) forms glucose and trehalose when allowed to act on substrate such as amylose and soluble starch. A purification of such as an enzyme preparation can not be completed, however, as the physicochemical properties of the enzyme thus prepared are not sufficiently indicated and the action of the enzyme has not been clarified, and only a preparation of trehalose is indicated. Thus, there has been a in great demand to establish a novel preparation of trehalose by utilizing a thermostable enzyme capable of acting at a temperature of over 55.degree. C.